B.nodosus is an anaerobic bacterium whose field isolates are characterized by the presence of surface filaments termed fimbriae (or common "Pili"), which are approximately 6 nm. in diameter and up to several hundred nm in length. In other bacteria, fimbriae have adhesive properties, and although the exact function of fimbriae in B.nodosus has not been clearly defined, it seems likely that they are involved in attachment to, and/or colonization of, epithelial tissues in the hoof. B.nodosus fimbriae have a polar location on the cell, and appear to be involved in surface translocation by a phenomenon known as twitching motility. Related fimbriae are found in a range of Gram-negative species classified within the genera Pseudomonas, Neisseria, Moraxella, Acinetobacter, Bacteroides and Eikenella, among others.
In the case of B.nodosus, a great deal of evidence has accumulated over the past decade to suggest that the fimbriae play a central role in both the infectivity of the bacteria and the protective immunological response of the sheep. Fimbriae are also implicated in pathogenesis and immunity in other bacterial pathogens. Vaccination of sheep with either whole cells or purified fimbriae from B.nodosus confers resistance to homologous footrot infections.
There are at least 8 or 9 major serogroups of B.nodosus (encompassing a number of subtypes), classified on the basis of the "K"-agglutination test.
The fimbriae have been shown to be the antigen involved in the specific agglutination reaction, and correspondingly the range of effective immunity conferred by vaccination with whole cells or fimbrial preparations is restricted to the serogroup/serotype involved.
The available commercial vaccines against footrot consist of a mixture of whole cells of different B.nodosus serotypes. However, the high cost of these vaccines, which is approximately an order of magnitude greater than that of other comparable vaccines (against e.g., the Clostridial group of infections), remains a serious obstacle to their widespread use and acceptance by the pastoral industry. As a result, present methods for the control of footrot continue to rely heavily on topical application of therapeutic agents, which have little or no impact on prevention of the disease.
The high cost of producing footrot vaccines is directly related to the difficulty of culturing fimbriate B.nodosus. This bacterium has very fastidious growth requirements, involving complex nutrient media and the absence of oxygen. Furthermore, since fimbrial expression is an unstable characteristic of B.nodosus, especially in the liquid broth culture conditions required for commercial production, batch-to-batch variation in the yields of fimbrial antigens may affect the quality and reliability of the vaccine preparations, introducing a significant cost burden in the area of quality control tests.
Applicants copending Australian patent application No. 34979/84 describes the production of the B.nodosus fimbrial subunit antigen expressed from cloned gene copies in Echerichia coli. The antigen is not assembled into mature fimbriae in these cells.
This invention describes the further development of this process, in that it describes the production of supramolecular, extracellular fimbrial structures from recombinant bacterial hosts. This provides substantial advantages in terms of both the antigenicity of the preparation (for vaccine purposes) and the practicalities and economics of antigen production. The invention makes use of a compatible fimbrial assembly system in a suitable Type 4 fimbrial host, such as Pseudomonas aeruginosa. This process is applicable to the production of fimbriae as vaccine antigens active against a variety of bacterial pathogens which produce Type 4 fimbriae.